Sterol regulatory element binding protein 1 (SREBP1) has a central regulatory effect on milk fat synthesis. Lipopolysaccharides (LPS) can induce mastitis and cause milk fat depression in cows. SREBP1 is also known to be associated with inflammatory regulation. Thus, in the current study, we hypothesized that LPS-induced milk fat depression in dairy cow mammary epithelial cells (DCMECs) operates via decreased SREBP1 expression and activity. To examine the hypothesis, DCMECs were isolated and purified from dairy cow mammary tissue and treated with LPS (10 µg/ml). LPS treatment of DCMECs suppressed lipid-metabolism-related transcription factor SREBP1 mRNA expression, nuclear translocation and protein expression, leading to reduced triglyceride content. The transcription levels of acetyl-CoA carboxylase-1 and fatty acid synthetase were significantly down-regulated in DCMECs after LPS treatment, suggesting that acetyl-CoA carboxylase-1 and fatty acid synthetase involved in de novo milk fat synthesis was regulated by SREBP1. In summary, these results suggest that LPS induces milk fat depression in dairy cow mammary epithelial cells via decreased expression of SREBP1 in a time-dependent manner.
Selenium (Se) is an important nutritional trace element possessing antioxidant properties. Our goal was to elucidate the effect and mechanism of Se deficiency on the intestinal cell fate. One-day-old three-yellow chickens were fed a low Se diet for 1, 3, and 5 weeks. Histologic characteristics, protein expression profiles, antioxidant activities, inflammatory signaling, and the apoptosis status in duodenum mucosa were investigated. Histological results showed that Se deficiency could increase inflammatory cell infiltration, karyopyknosis of the epithelial cells, cytoplasm vacuolization and dissolution of goblet cells. The proteomics results indicated that Se deficiency could induce apoptosis of cells in duodenal villi via inhibition of antioxidant redox signaling and activation of NF-κB signaling. Further analysis results showed that Se deficiency decreased the total antioxidant capacity of duodenum mucosa via down-regulating the transcription level and activities of glutathione peroxidase (GPX), reduced glutathione (GSH), and thioredoxin reductase (TrxR). The NF-κB signaling pathway was activated by Se deficiency-induced reactive oxygen species (ROS). TUNEL, DNA ladder, immunohistochemical assay, and western blotting proved that selenium deficiency could induce duodenal villi cell apoptosis. The results also indicated that Se deficiency can cause duodenal villi cell apoptosis via an oxidative stress-induced mitochondrial apoptosis pathway (intrinsic pathway) and an inflammatory signaling-induced death receptor pathway (extrinsic pathway). Our data may provide new insight into the prevention and treatment of chronic diarrhea caused by Se deficiency.
A set of four specific primers for six regions of kmt1 gene from a species specific region was designed for developing the loop-mediated isothermal amplification diagnostic method of swine Pasteurella multocida (Pm-LAMP). After the Pm-LAMP was carried out at 63°C for 1 h, the LAMP products could be visually confirmed using fluorescent dyes as detection reagent under UV-illumination. In sensitivity, the detection limit of the Pm-LAMP was 10 cfu/mL, and was 1 log less than that of the PCR method. In specificity, the Pm-LAMP did not amplify genomic DNA of swine common respiratory pathogens. Furthermore, based on results for clinical swab samples (n = 31) using PCR detection as golden standard, relative sensitivity of the Pm-LAMP was 100%, relative specificity of the Pm-LAMP was 90.9%, and percentage of observation agreement was 93.5% (Kappa = 0.85). The Pm-LAMP method should be a useful diagnostic tool for rapid and visible detection of swine Pasteurella multocida.
The entire pig aminopeptidase N (pAPN) gene was amplified by RT-PCR using total RNA extracted from intestinal brush border membrane of a newborn piglet. The amplified products of the pAPN gene were cloned into the vector pMD18-T, generating a recombinant plasmid pMD18-T-pAPN. The C subunit of pAPN (pAPN-C) produced by PCR from the plasmid pMD18-T-pAPN was expressed in Escherichia coli using vector pET-32a with His tag. After confirming reactivity of the recombinant protein pAPN-C to antibody against native pAPN, polyclonal antibody against the recombinant protein pAPN-C was prepared in rabbit using purified protein as immunogen. In Western blot analysis, the antibody elicited by the recombinant protein pAPN-C could recognize the native pAPN. These data demonstrate that the pAPN-C recombinant protein and its polyclonal antibody can provide some basis for further receptor antagonist.
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